Sirigiri Chandra Kala et al /2014
International Journal of Phytopharmacy
Research Article
Vol. 4 (1), pp.20-24, Jan-Feb 2014 ISSN: 2277-2928 (Online) Journal DOI:10.7439/ijpp ©Scholar Science Journals www.ssjournals.com
An efficient protocol devised for rapid callus induction from leaf explants of Biophytum sensitivum (Linn)DC. Sirigiri Chandra Kala1, Kokkanti Mallikarjuna1*, Patchala Aruna2 1
Department of Botany and Microbiology, Acharya Nagarjuna University, Nagarjuna Nagar, Guntur 522510, Andhra Pradesh, India. 2 Department of Microbiology, D.K. Govt. Degree College for Women, Nellore, Andhra Pradesh, India.
*Correspondence Info: Dr. Kokkanti Mallikarjuna, Department of Botany and Microbiology, Acharya Nagarjuna University, Nagarjuna Nagar, Guntur 522510, Andhra Pradesh, India. E-Mail:
[email protected]
Abstract The Cell cultures are used extensively for in vitro secondary metabolite productions were obtained from callus tissue through cell suspension culture. The establishment of callus cultures has considerable potential for the production of known and novel secondary metabolites. The objective of the study was to scientifically assess callus culture of Biophytum sensitivum (L) DC. This was established from leaf explants with different growth regulators which greatly influenced the growth of callus cultures. The callus from leaf explants is induced by inoculating the young leaf bits on MS medium supplemented with various auxins (2, 4Dichlorophenoxyacetic acid (2, 4-D), α-Naphthalene Acetic Acid (NAA) and Indole Buteric Acid (IBA), cytokinins (6-Benzyladenine (BA) Kinetin (KN) and cytokinin-auxin combination (BA+NAA) in different concentrations (0.5 to 5.0 mg/l) were used. BA 1mg/l, in combination with NAA (1.0 mg/l) also produced maximum amount of callus. So, this research concluded that the plant leaf explants cultured on MS medium with 1 mg/ l BA with 1 mg/l NAA was found most efficient for callus induction. Keywords: Biophytum sensitivum callus, leaf explants, callus induction and growth regulators
1. Introduction The Plant secondary metabolites have enormous potential for research and new drug development. The medicinal plants are the most important source of life saving drugs for the majority of the world population. A large number of medicinal plants are explored from the natural flora for the production of commercial drugs. It is a qualitative chemical evaluation, which indicates spectrum of chemical constituents present in a plant drug. Thus the phytochemical screening is very important in identifying new sources of therapeutically and industrially important compounds like alkaloids, flavonoids, phenolic compounds, saponins, steroids, tannins, terpenoids etc. 1 The qualitative analysis of the phytochemicals in the fresh plant tissue showed that phytochemical constituents such as alkaloids, anthroquinones, flavonoids, phenols, reducing sugars, saponins, phytosteroids and tannins are present in it. All the phyto components detected were known to support bioactive activities in medicinal plants. Approximately 20% of the plants found in the world have been submitted to pharmacological or biological tests. 2 The accumulation of phytochemicals in the plant cell cultures has been studied from the past thirty years, and the generated knowledge helped in realizing the usage of cell cultures for production of desired phytochemicals. 3 Plants extracts of secondary metabolites have served as antioxidant in phytotherapeutic medicines to protect against various diseases for centuries.4 So, cell suspension culturing is considered as one of the best approaches for studying the biosynthesis of natural products 5 and calli are the richest sources of cell mass when establishing such cultures. 1,6 Biophytum sensitivum common name is life plant, little tree plant and sensitive plant belongs to Oxalidaceae. Attapatti, Chumi, Jala pushpa in Telugu, Lakshmana, Lajalu in Hindi. It possesses a wide spectrum of medicinal properties including positive effects in inflammatory 7,8 diseases. It possesses a wide spectrum of medicinal properties namely antiseptic properties, asthma, and anti-inflammatory activity.9 The biological activity of the plant shows hypoglycemic,10 immunomodulatory11 effects. We previously reported that the in vitro pharmacological analysis using callus extracts of B.sensitivum.12 Cell cultures which have been used extensively for in vitro secondary metabolite production were obtained from callus tissue through cell suspension culture. So, further going to production of secondary metabolites using with different cytokinin and auxin combinations of using leaf explants.
2. Materials and Methods 2.1 Plant material: The fresh matured 100 plants of B.sensitivum were collected from Acharya Nagarjuna University Campus, Guntur District, Andhra Pradesh, India during 2009-2010 and used as a source of explants. The leaf explants were excised into 1 cm long segments and were washed with liquid detergent (Teepol, Qualigens, India), Bavistin (1% w/v) for 3 min and mercuric chloride (0.1% w/v) for 1 min, followed by 70% ethanol. These are inoculated on Murashige and Skoog medium (1962) 13 supplemented with various concentrations and combinations of phyto hormones for induction of callus. The combination of NAA+BA (BA 1 mg/l, NAA 1 mg/l) produced callus. 2.2 Callus culture: The leaf explants were cultured on MS basal medium supplemented with various concentrations of BA+NAA for callus induction. BA 1.0 mg/l + NAA 1.0 mg/l is the best concentration for callus induction. After 30-60 days, old callus was collected and sub cultured
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Sirigiri Chandra Kala et al /2014 on fresh medium with same growth regulator combinations twice in four week time interval. All the cultures were incubated at 24±2º C under 16h photoperiod provided by cool white florescent lights. 2.3 Data analysis: All the experiments were repeated thrice with 15 replicates. The effect of different treatments was analyzed using one way analysis of variance (ANOVA), and means were compared using the Tukey test at the 0.05 level of significance.
3. Results The callus from leaf explants is induced by inoculating the young leaf bits on MS medium supplemented with various auxins (2, 4-D, NAA and IBA), cytokinins (BA and KN) and cytokinin-auxin combinations (BA+NAA) in different concentrations (0.5 to 5.0 mg/l) (Table-1) were used. Results indicated that all the growth regulators alone not able to induce a callus from Biophytum sensitivum leaf explants. While the combinations of growth regulators show maximum callus production. Callus from leaf segments showed initiation of vigorous, proliferating, soft 14 and green colored tissue. The results indicated that 1.0 mg/l BA and 1.0 mg/l NAA (Fig 1A) on MS medium induced high amounts of callus with high frequency of regeneration interms of their fresh weight and dry weight (Table 1), (Fig 7). 2, 4-D (0.5 mg/l) with 3.0 mg/l BA (Table 1), (Fig 1 B, Fig 8), 2.0 mg/l IBA and 2.0 mg/l NAA (Table 1), alone BA 3mg/l (Fig 2), alone KN 0.5 mg/l (Fig 3), 2 mg/l of NAA and 0.5 mg/l 2,4-D was suitable for callus induction. The leaf explants when planted on the MS medium containing the combination of BA and NAA the young moderate sized leaf explants were well responded for rapid callogenesis after incubation period of about 3- 4 weeks. Green and healthy compact callus observed after 3-4 weeks of inoculation with 1 mg/l BA and 1 mg/l NAA combinations. Table 1: Effect of different concentrations of auxin and cytokinins on leaf callus induction Plant Growth Different concentrRegulators (mgL -1) ations (mgL -1) Control 0 0.5 1.0 2.0 BA 3.0 4.0 5.0 0.5 1.0 KN 2.0 3.0 4.0 5.0 0.5 1.0 IBA 2.0 3.0 4.0 5.0 0.5 1.0 NAA 2.0 3.0 4.0 5.0 0.5 1.0 2.4-D 2.0 3.0 4.0 5.0 0.5 1.0 1.0 1.0 BA+ NAA 2.0 1.0 3.0 1.0 4.0 1.0 5.0 1.0 0.5 0.5 BA+2,4-D 1.0 0.5 2.0 0.5 3.0 0.5 4.0 0.5 5.0 0.5
Nature of callus 0 Green callus, compact Green callus, compact Green callus, compact Green callus, compact Soft green Soft green Soft green Soft green Soft green Soft green Soft, green Soft, green Soft, green Green compact Green compact Green compact Green compact Light green, friable Light green, friable Light green, friable Light green, friable Green, compact Green, compact Green, compact Green, compact Green, friable Green, friable Green, friable Green, friable Green, friable -
Intensity of callus Results % of Fresh weight Dry weight formation response (mg)(SD) (mg) (SD) 0 0 0 0 0 + Callus 23 230 ± 1(21) 22 ± 3(53) + Callus 35 280 ± 1(32) 36 ± 2(10) ++ Callus 42 320 ± 2(14) 40 ± 2(41) +++ Callus 58 440 ± 1(83) 42 ± 2(12) No Callus No Callus ++ Callus 55 560 ± 1(41) 62 ± 3(53) + Callus 44 480 ± 1(32) 56 ± 2(10) + Callus 41 360 ± 2(12) 50 ± 2(41) + Callus 32 230 ± 1(82) 42 ± 2(12) + Callus 27 200 ± 1(21) 24 ± 2(14) + Callus 32 280 ± 1(32) 26 ± 1(11) + Callus 32 220 ±1(51) 22 ± 3(53) ++ Callus 45 340 ± 1(42) 36 ± 2(10) +++ Callus 58 520 ± 2(28) 40 ± 2(41) No Callus No Callus No Callus ++ Callus 43 420 ± 1(31) 22 ± 3(53) +++ Callus 57 580 ± 1(22) 36 ± 2(10) +++ Callus 58 660 ± 2(24) 40 ± 2(41) ++ Callus 42 230± 2(12) 24± 2(16) No Callus No Callus +++ Callus 70 440 ± 1(0) 58 ± 2(10) +++ Callus 65 360 ± 2(30) 50 ± 2(41) ++ Callus 52 240 ± 1(41) 32 ± 2(12) + Callus 47 200 ± 1(21) 24 ± 2(14) No Callus No Callus ++ Callus 52 320 ± 1(11) 28 ± 3(23) +++ Callus 82 1290 ± 11(26) 86 ± 2(25) ++ Callus 42 860 ± 2(46) 20 ± 2(21) + Callus 40 560 ± 1(12) 38 ± 2(10) No Callus No Callus + Callus 43 330 ±1(41) 12 ± 3(23) ++ Callus 55 450 ± 1(31) 26 ± 2(10) ++ Callus 57 470 ± 2(24) 30 ± 2(21) +++ Callus 68 670 ± 1(14) 48 ± 3(43) +++ Callus 65 530 ± 1(15) 52 ± 2(35) No Callus -
Values represent means ± standard error of 15 replicate per treatment in three repeated experiments. Mean followed by the same letter not significantly differently different by the Tukey Test at 0.05%. Intensity of callus: (+) low; (++) moderate; (+++) high.
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Sirigiri Chandra Kala et al /2014 Fig 1 (A) In vitro regenerated callus of Biophytum sensitivum after eight weeks of culture with BA 1.0 mg/l + NAA 1.0 mg/l. (B) Callus after eight weeks of culture with BA 3.0 mg/l + 2,4-D 0.5 mg/l
A
B
Fig 2: The callus growth variation with BA.
% of callus grow th
80
Control % of callus growth
60 40 20 0 0.5
1.0
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Fig 3: The callus growth variation with KN.
% of callus grow th
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Control % of callus growth 40
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0 0.5
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Fig 4: The callus growth variation with IBA.
% of callus growth
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Control % of callus growth
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Sirigiri Chandra Kala et al /2014 Fig 5: The callus growth variation with NAA.
% of callus growth
60
Control % of callus growth 40
20
0 0.5
1.0
2.0
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Fig 6: The callus growth variation with 2, 4-D.
% of callus growth
80
Control % of callus growth
60 40 20 0 0.5
1.0
2.0
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2,4-D concentrations(mgL-1)
Fig 7: The callus growth variation with BA+NAA.
% of callus growth
80
Control % of callus growth
60 40 20 0 0.5
1.0
2.0
3.0
4.0
5.0
BA+NAA concentrations(mgL -1)
Fig 8: The callus growth variation with BA+2,4-D.
% of callus growth
80
Control % of callus growth
60 40 20 0 0.5
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2.0
3.0
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BA+2,4-D concentrations(mgL-1)
4. Discussion Callus tissue is a good source of genetic variability and adventitious shoot formation. Regeneration of plants from callus tissue by organogenesis or somatic embryogenesis is an alternative method of shoot multiplication for cloning plant species.15 Biophytum sensitivum is most important medicinal plant widely used in indigenous systems of medicine in India. Callus tissue was not uniform. Both friable and compact calli were obtained. Some were of fine texture and some were of nodular. These differences can be attributed to various factors like type of the explant, constituents of the medium and cultural environment. This is because of cells in callus cultures are undifferentiated and may not be under specific control. The auxin and cytokinin ratio proved their importance for callusing in various explants. Further increase in the concentration of plant growth regulators did not show any significant improvement in callusing. These results agree with induced callus from the leaves of Solanum 16 tuberosum. Although cell cultures offer a suitable biological system in a controlled environment where in the morphogenic events can be maintained and regulated by growth regulators in the nutrient medium which shows a rapid production of plant metabolites of pharmaceutical value.17
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Sirigiri Chandra Kala et al /2014 5. Conclusion In the present study, in vitro technique was chosen to develop and conserve this medicinally important plant for future. Phytochemical constituents are the basic source for the establishment of several pharmaceutical industries. The constituents present in a drug or plant play a significant role in the identification of crude drug.
Acknowledgements The first author Sirigiri Chandra Kala is thankful to University Grants Commission, New Delhi for providing financial support with Rajiv Gandhi National Fellowship.
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